The immediate goal of this project is to define the cellular and biochemical functions of 57 uncharacterized open reading frames (ORFs), hypothetical proteins, and small non-coding RNAs in B. abortus. Our preliminary data provide evidence that these genes have a functional role in general stress adaptation and chronic mammalian infection. General stress response (GSR)-dependent transcription in B. abortus is controlled by the sigma factor, SigEI. Our preliminary investigation of SigEI and its upstream signaling partner, PhyR have demonstrated that these regulatory proteins are required for adaptation to oxidative and acid stress in vitro, and for maintenance of chronic infection in a BALB/c murine model. We have experimentally defined a set of 108 genes that are directly or indirectiy regulated by SigEI. Within this regulated gene set are 32 uncharacterized open reading frames (ORFs) and 9 small non-coding RNAs. Additionally, we have identified 16 hypothetical genes that are adjacent to, or in apparent operons with SigEI-regulated ORFs, but for which we have no evidence of expression. We propose to: 1) Test strains in which these genes have been deleted in oxidative and acid stress survival assays, and in cell-based and animal infection assays, 2) elucidate the biochemical functions of these hypothetical proteins and SigEI-regulated ORFs in collaboration with out Biochemical Function Technology Core, and 3) characterize the biochemical function of putative non-coding RNAs under transcriptional control of SigEI. These experiments will provide a comprehensive (in vivo and in vitro) functional understanding of conserved and non-conserved B. abortus genes that are currently uncharacterized.